1. Field of Invention
The present invention relates to hot electron-operated electrochemical reactors which are applied to carry out chemical reactions. The device comprises working electrode/electrodes which are coated according to the prior art with an electrically insulating layer, which enables to carry out non-conventional electrochemical redox reactions. The electrode device has a wide area of application as a chemical reactor, in analytical chemistry, and in biochemistry as a sensitive detector, in particular, with methods based on biorecognition, such as in clinical diagnostics.
2. Description of the Related Art
A considerable improvement over the active metal or semiconductor electrodes used to carry out different electrochemical reactions was provided by the publication WO 98/36266 (U.S. Pat. No. 6,251,690 Kulmala et al.). These so-called insulating film-coated electrodes can be applied to the generation of exceptional chemical reactions at high redox energy levels. When such electrode reactions are effected in the presence of certain organic or inorganic luminophores, light is generated. Whereas a variety of applications based on this phenomenon can be readily advised, one of the most evident one is the use of such a system as a sensitive detector in analytical chemistry as described in WO 98/36266.
Many commercially important analytical methods require sensitive and specific detectors. The majority of such methods are based on the principle that the analytes are separated by their biological function and thereafter quantified using certain label substances. For instance, in the assays based on the biological properties of analytes, such as in immunoassays, the analyte (A) can be selectively captured from a solution upon a solid support with the aid of antibodies immobilized on the surface of the solid support, and the amount of (A) can be quantified using another antibody selectively binding with (A) and being labeled with a suitable marker substance. Such a marker substance can be, for instance, a radioactive isotope, an enzyme, a molecule that absorbs light or produces fluorescence or phosphorescence, certain metal chelates etc., which can be coupled with an antibody via chemical bonds. Alternatively, purified (A) can be labeled (A-L) and the amount of unlabeled (A) can be determined by antibodies immobilized on a solid support by exploiting a competitive reaction between (A-L) and analyte (A). DNA- and RNA-probing assays are based on analogous bioaffinity principles to those of immunoassays and can be performed along with related procedures. Also, other chemical and biochemical analysis methods can be based on analogous principles. Presently, there is an increasing need for multiparameter assays due to a growing demand to decrease the costs and/or increase the simplicity and accuracy of determinations. One solution to these problems is the use of label compounds which luminesce at different wavelengths. Various methods and strategies in immunoassays are described, e.g., in “The Immunoassay Handbook”, Edited by David Wild, Stockton Press Ltd., New York, 1994, pages 1–618.
Totally new, strongly commercially emerging technologies are also based on the exploitation of biorecognition. This is called DNA microarray technology and has attracted tremendous interests among biologists. It is widely known that thousands of genes and their products (i.e. RNAs and proteins) in a given living organism function in a complicated and orchestrated way. Traditional methods of molecular biology generally work on a “one gene in one experiment” basis, which means that the “whole picture” of a gene function is hard to obtain. The new DNA microarray technology promises to monitor the whole genome on a single chip so that interactions among thousands of genes can be investigated simultaneously. Terminologies that have been used in the literature for such a device, include, but are not limited to: biochip, DNA chip, gene chip, DNA microarray, gene array, and genome array. For RNA and proteins, the related techniques are called RNA microarray and proteomics, respectively. The commercial applications of DNA microarray technology include gene discovery, disease diagnostics (commercially the most important), drug discovery, and toxicology.
The present technical level of the microarray technologies are exemplified by the following articles: M. Schena et al., Trends in Biotechnology, 1998, 16, 301–306; G. MacBeath and S. Schreiber, Science, 200, 289(5485), 1760–1763; and G. Ramsay, Nature Biotechnology, 1998, 16, 40–44.
These new technologies demand extremely high sensitivities and flexibilities of the detector systems, while at the same time the dimensions must be miniaturized to an utmost minimum.
It is already known that organic luminophores and metal chelates suitable for labeling in analytical methods can be excited with light or by electrochemical means resulting in the specific emission of light from the labeling substance. The methods based on these phenomena are generally sensitive and well-suited for the excitation of label substances. However, difficulties are encountered when the concentrations of labels in real assays are very low; e.g., the use of fluorescence is complicated by the existence of Tyndall, Raleigh, and Raman scattering, and by the background fluorescence common in biological samples. Phosphorescence in liquid phase is mainly usable only in connection with some specially synthesized lanthanide chelates. Utilization of the long-lived photoluminescence of these compounds is restricted mainly due to the complicated apparata required and high cost of pulsed light sources.
Electrochemiluminescence can be generated in non-aqueous solvents at inert metal electrodes with a rather simple apparatus. However, certain chemical reactions like bioaffinity assays which are of commercial importance are normally applicable in aqueous solutions only. Samples are practically always aqueous and therefore the detection method of a label substance must be applicable in aqueous solution. Presently, only certain transition metal chelates can serve as electrochemiluminescent labels in micellar solutions, which, in fact, are to be considered at least partly as non-aqueous solutions. However, these methods utilizing conventional electrochemistry and inert metal electrodes do not allow simultaneous excitation of several label substances possessing sufficiently differing emission spectra and/or luminescence lifetime.
Mainly inert active metal electrodes are applied in conventional electrochemistry. Their utilization is restricted to a narrow potential window due to the water decomposition reactions, resulting in hydrogen and oxygen evolution. Luminophores usable as fluorescent or phosphorescent labels cannot normally be electrically excited in aqueous solution at these electrodes due to the inaccessibility of the highly anodic and cathodic potentials required for the excitation reactions. With suitably selected semiconductor electrodes, a wider potential window is achievable, but only very rare labeling substances can be excited at these type of electrodes in fully aqueous solutions. A considerable improvement for the use of active metal electrodes or semiconductor electrodes was provided by WO 98/36266 (Kulmala et al.) which made it possible to simultaneously excite a variety of different labeling substances in a fully aqueous solution. This invention by Kulmala et al. utilized a new type of electrochemistry and electrodes, conductors covered with a thin insulating film, which cannot be used in conventional electrochemistry. These electrodes are called insulator electrodes or insulating film-coated electrodes. According to the present invention, is was suprisingly observed that their performance and applicability is considerably improved by constructing a channeled texture of the electrode surfaces.
As a technical construction, two patent publications, WO 99/09042 and WO 99/33559 describe microfabricated devices used for chromatographic separation and concentration of different substances. Although certain technical features, like micropores or channels, disclosed in said publications resemble those used in the present invention, their meaning and applications are completely different. In the present invention, microstructures are created on electrode devices to be used for carrying out electrogenerated chemical reactions in the vicinity of the electrode surfaces based on the hot electron-induced mechanism. The main application of the electrode device of the present invention is its use as an electrochemical detector with a photomultiplier tube or photodiode. In the future, various other applications based on this novel electrochemistry will be expected. A drawback of utilizing the insulating film-coated electrodes disclosed in WO 98/36266, was the relatively long assay times due to the macro-scale cells employed and the low specific surface areas of the working electrodes, which affects the sensitivity and the applicability of such electrodes. In theory, the insulating film-coated electrodes work in relatively simple systems. However, the electrodes, as described in the prior art, are useful only for limited applications and do not fullfil the requirements of the most modem trends in the development of analytical chemistry. The electrodes are not sensitive enough for the most demanding modern applications and they are not essentially suitable for functioning as flow-through detectors because they were mainly aimed at application to batch assays. The most severe drawback when using said electrodes is the very high diffusion times of the biorecognition reactions on the macro-scale electrodes. Further, although the simple insulator electrodes in theory have certain definite advantages, their development was not satisfactory for practical purposes from technological, design, and manufacturing points of view. Especially this is true in the context of large scale production.
The above-mentioned drawbacks can be avoided by using the new electrode constructions according to the present invention. It was found surprisingly that channeled micromachined or etched insulator electrodes give significantly higher performance while allowing fast bioreactions to occur at the novel electrodes of this invention. The channeled electrodes described in the present invention have several advantages over the prior art.